We have been involved for many years in the study of a group of proteins that tightly bind members of the folic acid family of coenzymes and have discovered four such proteins that have new and unusual functions. Two of these are enzymes that were found to be present in liver mitochondria, dimethylglycine dehydrogenase (DMGDH) and sarcosine dehydrogenase (SDH). Over 25 years ago we showed that both enzymes carry out the removal of methyl groups that are bound directly to a carbon atom. We showed the mechanism of demethylation to be the initial oxidation of the methyl-amine bond in dimethylglycine to the imine followed by the hydrolysis of the imine to the demethylated product plus formaldehyde derived from the methyl group. The function of the folate coenzyme, tetrahydrofolate pentaglutamate (THF-G5), is to scavenge the formaldehyde that is generated by converting it to 5,10-methylene-tetrahydrofolate (5,10-CH2 because formaldehyde is toxic and can cross-link proteins. A recently discovered family of histone demethylases catalyzes histone demethylation by the same mechanism as DMGDH. The best characterized member of this family is histone lysine demethylase 1 (LSD1). Study of the participation of THF-G5 in histone demethylation is the focus of our project. Our First Hypothesis is that THF-G5 acts as a scavenger of enzymatically generated formaldehyde in the histone demethylases. Our First Specific Aim is to determine whether THF-G5 binds to histone lysine demethylase-1 (LSD-1), the first member of this family for which the mechanism of the reaction was studied. Our Second Hypothesis is that the THF-G5 bound to LSD-1 is converted to 5,10-CH2-THF-G5 during the reaction to protect the enzyme from the toxic formaldehyde generated during the reaction. Our Second Specific Aim is to isolate the 5,10-CH2-THF-G5 formed during the reaction. Our Third Hypothesis is that THF-G5 is situated in the molecules of both DMGDH and LSD1 to permit the formation of an adduct between the formaldehyde and THF-G5. Our Third Specific Aim is to crystallize each enzyme in the presence of THF- G5 and compare the protein-ligand interactions. PUBLIC HEALTH RELEVANCE: This proposal is a continuation of our investigation into the function of a group of folate-binding proteins that we discovered and showed are enzymes that carry out new and previously unknown functions. A major goal is to investigate a possible new class of folate binding-proteins that are involved with histone demethylation and regulate protein expression. This provides a new role for the family of folic acid derived coenzymes in the nucleus.